Significance: The initial dip in hemoglobin-oxygenation response to stimulations is a spatially confined endogenous indicator that is faster than the blood flow response, making it a desired label-free contrast to map the neural activity. A fundamental question is whether a single-impulse stimulus, much shorter than the response delay, could produce an observable initial dip without repeated stimulation.
Aim: To answer this question, we report high-speed functional photoacoustic (PA) microscopy to investigate the initial dip in mouse brains.
Approach: We developed a Raman-laser-based dual-wavelength functional PA microscope that can image capillary-level blood oxygenation at a 1-MHz one-dimensional imaging rate. This technology was applied to monitor the hemodynamics of mouse cerebral vasculature after applying an impulse stimulus to the forepaw.
Results: We observed a transient initial dip in cerebral microvessels starting as early as 0.13 s after the onset of the stimulus. The initial dip and the subsequent overshoot manifested a wave pattern propagating across different microvascular compartments.
Conclusions: We quantified both spatially and temporally the single-impulse-stimulated microvascular hemodynamics in mouse brains at single-vessel resolution. Fast label-free imaging of single-impulse response holds promise for real-time brain–computer interfaces.
Large-scale, high-resolution imaging of cerebral hemodynamics is essential for brain research. Uniquely capable of comprehensive quantification of cerebral hemodynamics and oxygen metabolism in rodents based on the endogenous hemoglobin contrast, multiparametric photoacoustic microscopy (PAM) is ideally suited for this purpose. However, the out-of-focus issue due to the uneven surface of the rodent brain results in inaccurate PAM measurements and presents a significant challenge to cortex-wide multiparametric recording. We report a large-scale, high-resolution, multiparametric PAM system based on real-time surface contour extraction and scanning, which avoids the prescan and offline calculation of the contour map required by previously reported contour-scanning strategies. The performance of this system has been demonstrated in both phantoms and the live mouse brain through a thinned-skull window. Side-by-side comparison shows that the real-time contour scanning not only improves the quality of structural images by addressing the out-of-focus issue but also ensures accurate measurements of the concentration of hemoglobin (CHb), oxygen saturation of hemoglobin (sO2), and cerebral blood flow (CBF) over the entire mouse cortex. Furthermore, quantitative analysis reveals how the out-of-focus issue impairs the measurements of CHb, sO2, and CBF.
In the current form of multi-parametric photoacoustic microscopy (PAM), imaging hemoglobin concentration and blood flow speed requires dense sampling. Moreover, large-scale recording beyond the focal zone of ultrasonic transducer requires time-consuming mechanical scan of the optical-acoustic dual foci. Thus, the image acquisition time of multi-parametric PAM has been severely limited by the laser repetition rate and the focal diameter of the transducer.
Here, we report an ultrahigh-speed multi-parametric PAM with 1.2-MHz A-line rate for simultaneous real-time imaging of hemoglobin concentration, blood oxygenation, and blood flow in the mouse brain. Capitalizing on the pronounced stimulated Raman scattering in pure silica-core polarization-maintaining single-mode optical fibers, a dual-wavelength (532 and 558 nm) nanosecond laser with 1.2-MHz pulse repetition rate has been developed. Using a weakly focused ultrasonic transducer, we have achieved real-time acquisition of multi-parametric PAM images at a frame rate of 2.2 Hz over the 250-μm-diameter acoustic focal zone. By employing optical-mechanical hybrid scan, 25 dual-wavelength B-scans can be acquired simultaneously within one mechanical-scan trip, leading to a 25-fold improvement of imaging
speed. As a result, the imaging frame rate is improved from 0.08 Hz in the conventional multi-parametric PAM to 2.2 Hz.
The utility of this new PAM technology has been demonstrated in a mouse model of epilepsy by studying the dynamic neurovascular uncoupling during status epilepticus.
General anesthetics are known to have profound effects on cerebral hemodynamics and neuronal activities. However, it remains a challenge to directly assess anesthetics-induced hemodynamic and oxygen-metabolic changes from the true baseline under wakefulness at the microscopic level, due to the lack of an enabling technology for high-resolution functional imaging of the awake mouse brain. To address this challenge, we have developed head-restrained photoacoustic microscopy (PAM), which enables simultaneous imaging of the cerebrovascular anatomy, total concentration and oxygen saturation of hemoglobin (CHb and sO2), and blood flow in awake mice. From these hemodynamic measurements, two important metabolic parameters, oxygen extraction fraction (OEF) and the cerebral metabolic rate of oxygen (CMRO2), can be derived. Side-by-side comparison of the mouse brain under wakefulness and anesthesia revealed multifaceted cerebral responses to isoflurane, a volatile anesthetic widely used in preclinical research and clinical practice. Key observations include elevated cerebral blood flow (CBF) and reduced oxygen extraction and metabolism.
Enabling simultaneous high-resolution imaging of the total concentration of hemoglobin (CHb), oxygen saturation of hemoglobin (sO2), and cerebral blood flow (CBF), multiparametric photoacoustic microscopy (PAM) holds the potential to quantify the cerebral metabolic rate of oxygen at the microscopic level. However, its imaging speed has been severely limited by the pulse repetition rate of the dual-wavelength photoacoustic excitation and the scanning mechanism. To address these limitations, we have developed a new generation of multiparametric PAM. Capitalizing on a self-developed high-repetition dual-wavelength pulsed laser and an optical–mechanical hybrid-scan configuration, this innovative technique has achieved an unprecedented A-line rate of 300 kHz, leading to a 20-fold increase in the imaging speed over our previously reported multiparametric PAM that is based on pure mechanical scanning. The performance of the high-speed multiparametric PAM has been examined both in vitro and in vivo. Simultaneous PAM of microvascular CHb, sO2, and CBF in absolute values over a ∼3-mm-diameter brain region of interest can be accomplished within 10 min.